The present invention relates generally to methods for the treatment and prevention of diabetes and to the use of cells which can express insulin in a glucose-regulated manner for this treatment.
In an individual with normal regulation of blood glucose, the pancreatic hormone insulin is secreted in response to increased blood sugar levels. Increased blood glucose generally occurs following a meal and results from insulin action on peripheral tissues such as skeletal muscle and fat. Insulin stimulates cells of these peripheral tissues to actively take up glucose from the blood and convert it to forms for storage. This process is also referred to as glucose disposal. The levels of blood glucose vary from low to normal to high throughout the day within an individual, depending upon whether the person is in the fasting, intermediate, or fed state. These levels are also referred to as hypoglycemia, euglycemia and hyperglycemia, respectively. In the diabetic individual, these changes in glucose homeostasis are disregulated due to either faulty insulin secretion or action, resulting in a chronic state of hyperglycemia.
Diabetes mellitus is a common disorder, with a prevalence of about 4-5%. The risk of developing diabetes increases with increased weight, with as many as 90% of adult onset diabetic patients being obese. Therefore, due to the high incidence of obese adults, the incidence of adult onset diabetes is increasing worldwide. Diabetes mellitus is classified into three major forms. Type 2 diabetes is one form and is also referred to as non-insulin dependent diabetes (NIDDM) or adult-onset diabetes. Type 1 diabetes is the second form and is referred to as insulin-dependent diabetes (IDDM). The third type of diabetes is genetic and is due to mutations in genes controlling pancreatic islet beta (xcex2) cell function. Although the diagnosis of diabetes is based on glucose measurements, accurate classification of all patients is not always possible. Type 2 diabetes is more common among adults and type 1 diabetes dominates among children and teenagers.
Diabetes mellitus of both types 1 and 2 are associated with a shortened life expectancy as well as other complications such as vascular disease and atherosclerosis. Long-term management of diabetes to prevent late complications often includes insulin therapy regardless of whether the patients are classified as type 1 or type 2. Type 1 diabetes is an auto-immune disease which is associated with near complete loss of the insulin producing pancreatic xcex2 cells. This loss of xcex2 cells results in insulin-dependence for life. Type 1 diabetes can occur at any age and it has been estimated that about 1% of all newborns will develop this disease during their lifetime.
Insulin is first synthesized as a precursor and cleaved through the action of proteases to form a mature, bioactive molecule that consists of two subunits covalently bonded together. Insulin function and regulation have been studied in regard to both therapeutic and research applications. For example, insulin has been produced for therapeutic purposes by recombinant expression using modified cells. As a research tool, insulin was the first protein to be chemically synthesized and also has been used to study secretory pathways and the regulation of secretory mechanisms.
A widely used method of treatment for type 1 diabetes and to some extent type 2 diabetes has classically consisted of insulin maintenance therapy. Such therapy in its simplest form requires the injection of purified or recombinant insulin into a patient following ingestion of a meal or at regular intervals throughout the day to maintain normal blood glucose levels. These injections are required ideally at a frequency of four times per day. Although the above method of treatment provides some benefit to the patient, this method of insulin therapy nevertheless suffers from inadequate blood glucose control as well as requiring a great deal of patient compliance.
Another method of treatment for type 1 diabetes includes the use of devices such as an insulin pump which allows for the scheduled delivery of insulin. This method can be preferable to the method described above due to the need for less frequent injections. However, the use of an insulin pump therapy also has drawbacks in that replacement of a needle once every three days is still required. Similar to insulin maintenance therapy, the insulin pump method also does not achieve optimal glucose regulation as the delivery of insulin is not regulated in response to changes in blood glucose level. These methods of treating diabetes are therefore burdensome as well as inadequate. Furthermore, although these methods can provide some benefit for reducing symptoms of diabetes, none have been completely effective over the course of an average adult lifetime and none have been shown to be effective in preventing this disease.
Various approaches of cell therapy for replacing bioactive insulin into a diabetic individual have been attempted. These include gene therapy approaches, immunotherapies and use of artificial xcex2-cells.
In vivo gene therapy for insulin expression has included liver targeted retroviral-mediated transduction in rats and adenoassociated virus administration to mice (Kolodka et al., Proc. Natl. Acad. Sci. USA 92:3293-3297 (1995); Sugiyama et al., Horm. Metab. Res. 29:599-603 (1997)). However, these approaches did not provide glucose-regulated insulin delivery and have limited applications in patients.
Treatment for diabetes has also included studies attempting to manipulate the immune system. In particular, to reverse the autoreactivity against the beta cell specific autoantigens, insulin or glutamic acid decarboxylase (GAD65) have been injected into young diabetes-prone NOD mice or BB rats (Kaufman et al., J. Clin. Invest. 89:283-292 (1992); Kaufman et al., Nature 366:69-72 (1993); Bieg et al., Diabetologia 40:786-792 (1997)). Reversal of autoreactivity against beta cell specific autoantigens has also been attempted with nasal administration of GAD65 peptides (Tian et al., J. Exp. Med. 183:1561-1567 (1996)).
Genetic modification of pancreatic islet xcex2-cells and generation of artificial beta cells are approaches for the treatment of diabetes by cell therapy (Becker et al., Methods Cell Biol. 43:161-189 (1994); Efrat, Diabetes Reviews 4:224-234 (1996); Newgard, Diabetes 43:341-350 (1994); Gros et al., Hum. Gene Ther. 8:2249-2259 (1997); Taniguchi et al., J. Surg. Res. 70:41-45 (1997). Xenograft and even allogeneic cell delivery to express insulin require cell encapsulation to prevent host immune responses, and problems with cell survival and sustained insulin delivery have been identified (Kawakami et al., Cell Transplant. 6:541-545 (1997); Wang et al., Nature Biotechnol. 15:358-362 (1997); Zhou et al., Am. J. Physiol. 274:C1356-C1362 (1998); Scharp et al., Diabetes 43:1167-1170 (1994)).
Pancreatic and islet transplantation has also been attempted as a treatment for diabetes. Use of this treatment has shown limited success due to the requirement for matched tissue from 2-5 adult donors per recipient. This method has also lacked success due, in part, to the failure of the transplanted tissue to maintain normal glucose-regulated insulin secretion and to remain viable over a reasonable period of time.
Thus, there exists a need for simple and more efficient methods that can regulate glucose homeostasis in a diabetic individual in a way that more closely mimics a normal endogenous insulin response. The present invention satisfies this need and provides additional advantages.
The present invention provides an isolated population of cells containing an expressible nucleic acid encoding proinsulin containing a proinsulin cleavage site and a glucose-regulated expressible nucleic acid encoding a protease capable of cleaving the proinsulin cleavage site to produce insulin. The invention also provides an isolated population of cells which further express a hexosamine synthetic pathway enzyme. The invention additionally provides vectors containing an expressible nucleic acid encoding proinsulin containing a proinsulin cleavage site and a glucose-regulated expressible nucleic acid encoding a protease capable of cleaving the proinsulin cleavage site to produce insulin. The invention further provides a method of treating or preventing diabetes by implanting into an individual cells coexpressing proinsulin containing a proinsulin cleavage site and a glucose-regulated protease capable of cleaving the proinsulin cleavage site to produce insulin.